Several decades ago, prolamins were described as very specialized storage proteins involved in the formation of natural protein bodies (PB) in the endosperm of cereals. Sherry et al., 1990, Biochem. J. 267:1-12. Nevertheless, to this day, little is known about the requirements for the formation of this type of organelle.
The endosperm is a specialized plant tissue that appears to have a greater tendency to sort proteins into PBs than other tissues and cell types. This is true even when the proteins are not fused to prolamins. For example, recombinant phytase protein, which is secreted from rice leaf cells, is retained in PBs when it is expressed in the endosperm. Drakakaki et al., 2006, Plant Physiology, 141, 578-586. Similarly, the major glycoprotein (gB) of human cytomegalovirus (Wright et al., 2001, Transgenic Research 10: 177-181) and lysozyme (Yang et al., 2003, Planta 216: 597-603) also accumulate in PBs in dicot and monocot plant endosperms even when they are not fused to prolamins. Interestingly, even when lysozyme was fused to a non-prolamin related signal peptide and expressed under the control of a non-prolamin promoter (puroindoline b) it accumulated in PBs in rice endosperm. Hennegan et al., 2005, Transgenic Research 14:583-592. These data suggest that accumulation of proteins in PBs could require the specialized storage environment of the endosperm.
The hightened propensity for proteins to be sorted in endosperm is also demonstrated by experiments using KDEL-(SEQ ID NO:162) tagged recombinant proteins. The KDEL tag is also known as an “ER retention signal” because it helps maintain proteins in the endoplasmic reticulum (ER). Thus, when human serum albumin is fused to the KDEL tag, it localizes to the ER lumen in leaf cells. However, when expressed in the endosperm, KDEL-tagged human serum albumin was deposited in prolamin aggregates within vaculoes. In addition, a KDEL-tagged monoclonal antibody, which is efficiently retained in the endoplasmic reticulum in leaves, was partially secreted and partially sorted to protein storage vacuoles in seeds. Petruccelli et al, 2006, Plant Biotechnol J. 4:511-27.
Therefore, protein transport within cereal endosperm cells is affected by the endosperm-specific environment, including the abundance of endoplasmic reticulum (ER)-derived and vacuolar protein bodies. Accordingly, proteins that are sorted to PBs in the endosperm may not be sorted there in other cells or tissues, and sequences and structures that are sufficient to induce formation of PBs in the endosperm may not necessarily be sufficient for formation of PBs in other cells or tissues.
Furthermore, the specific sequences and structures that are sufficient to induce formation of PBs have not been identified. In fact, when all of the proteins involved in PB formation are compared, no clear homology in terms of sequence, structure, or physical and chemical characteristics is evident.
Gamma zein is a major constituent of protein bodies in maize. Ludevid et al., 1984, Plant Mol. Biol. 3: 227-234. The N-terminal domain of gamma zein contains a Pro-X region (P-X) and a highly repetitive sequence (PPPVHL)6(PPPVHV)(PPPVHL) (repeat domain; “RD”) (SEQ ID NO:158) necessary for sorting gamma zein in the ER (Geli, et al. Plant Cell 6:1911 (1994)) and for the formation of protein bodies. See also U.S. Published Application No. 2007/0243198. A circular dichroism study of a synthetic peptides series of the sequences (VHLPPP)×3 (SEQ ID NO:159), (VHLPPP)×5 (SEQ ID NO:160), and (VHLPPP)×8 (SEQ ID NO:161) in water at pH 5, showed that these peptides adopt a polyproline II (PPII) helix (Rabanal, Biopolymers 33: 1019-28 (1993)). Gamma zein also contains several cysteines that were shown to be required for the formation of stable PBs. Pompa, Plant Cell 18: 2608-2621 (2006).
The PPII helix of the RD of gamma zein has a marked amphipathic character. Previous studies have suggested that the amphipathic nature of the PPII helix was important for the formation of stable PBs, and the surfactant properties of the amphipathic PPII helix (VHLPPP)×8 (SEQ ID NO:161) have been demonstrated by several approaches. Kogan et al., 2001, J. Mol. Biol. 312: 907-913003; Kogan et al., 2002, Biophysical J 83: 1194-1204. For example, it was shown that the synthetic octamer peptide (VHLPPP)×8 (SEQ ID NO:161) was able to lower the surface tension of water, due largely to the adsorption of the amphiphile to the air-water interface with the hydrophobic moiety oriented away from the aqueous phase. Ludevid et al., 1984, Plant Mol. Biol. 3: 227-234. It was also demonstrated that this amphipathic peptide interacts with soybean phosphatidylcholine liposomes and assembles to form extended domains over the membrane, increasing its stability and permeability. Kogan et al., 2004, Biopolymers, 73: 258-268. The spontaneous amphipathic assembly of (VHLPPP)×8 (SEQ ID NO:161) on the membrane suggests a mechanism of gamma-zein deposition inside maize protein bodies. Based on the amphipathic characteristics of gamma-zein RD, it has been proposed that this protein interacts with the inner face of the ER membrane inducing an internal coat that could be a key element in the mechanism of PB induction (Ludevid, 1984). This coating may then be covalently stabilized via intramolecular disulfide cross-linking involving cysteine residues that flank the repetitive sequence of gamma zein.
While some of the features of the gamma-zein protein have been characterized, it was not previously understood which of these features or combination of features was relevant for protein body formation. Furthermore, other protein body-inducing sequences contain little or no structural or sequence similarity to gamma-zein. As described in more detail below, a minimum polypeptide capable of inducing protein bodies has been identified. Furthermore, recombinant protein body-inducing sequences with improved properties, such as an increased ability to form recombinant protein body-like assemblies (RPBLAs) and an ability to form RPBLAs with improved characteristics, have been identified.